人MxA基因重组真核载体抗乙型肝炎病毒作用的实验室研究

发布时间：2018-05-21 18:20

本文选题：MxA + PcDNA3.1　；参考：《重庆医科大学》2007年硕士论文

【摘要】： 第一部分人MxA基因重组真核载体的构建及鉴定目的:在前期获得含有目的基因MxA的PAdtrack-MxA(含MxA的重组腺病毒穿梭质粒)基础上,应用真核载体PcDNA3.1构建重组真核载体PcDNA3.1-MxA。方法:在大肠杆菌JM109中扩增获得含有目的基因MxA的PAdtrack-MxA和PcDNA3.1,使用两者共同的限制性内切酶Xba I、Not I进行双酶切,并连接以构建重组真核载体PcDNA3.1-MxA,然后通过氨苄青霉素抗性筛选,双酶切及PCR鉴定,选取鉴定正确的克隆测序,并应用DNAssist 2.0软件对序列与前期获得的基因序列以及已知GenBank中MxA基因序列进行同源性分析。结果:经限制性内切酶、PCR鉴定重组真核载体PcDNA3.1-MxA构建成功。测序结果表明本实验所克隆片段长2012bp,包含人MxA基因序列,核苷酸序列分析表明与前期获得的目的基因序列完全相同,而与GenBank中人MxA基因序列同源性也达99.75%,仅有5个碱基不同,且所编码的氨基酸仅1个发生改变,此氨基酸位于MxA蛋白的非功能区。结论:重组真核载体PcDNA3.1-MxA构建成功,为下一步研究MxA抗乙型肝炎病毒作用奠定了基础。第二部分人MxA基因重组真核载体抗乙型肝炎病毒作用的实验室研究目的:体外研究PcDNA3.1-MxA体外抗HBV的作用,为进一步研究体内抗病毒及临床应用提供理论和实验基础。方法:将构建成功的重组真核载体PcDNA3.1-MxA转染入HepG 2.2.15,根据PcDNA3.1-MxA新霉素抗性,使用G418抗性筛选出稳定的细胞克隆。将HepG 2.2.15分为实验组(稳定表达PcDNA3.1-MxA的HepG 2.2.15)和对照组(未转染组),应用RT-PCR方法检测两组HepG2.2.15内MxA mRNA水平的表达,并应用ELISA法检测两组HepG2.2.15内HBsAg、HBeAg的水平。结果:经浓度梯度筛选显示G418最适筛选浓度为600μg/ml,并在培养第3周筛选出稳定表达PcDNA3.1-MxA的HepG 2.2.15。RT-PCR扩增结果显示实验组的MxA mRNA水平均较对照组明显升高,而且ELISA检测结果显示实验组HBsAg、HBeAg水平明显低于对照组,其结果具有显著的统计学意义(P㩳0.01)。结论:在HepG 2.2.15内成功转染并稳定表达重组真核载体PcDNA3.1-MxA,PcDNA3.1-MxA能抑制HepG 2.2.15中HBV的复制及表达。
[Abstract]:Construction and Identification of Recombinant Eukaryotic Vector of Human MxA Gene Aim: to construct the recombinant eukaryotic vector PcDNA3.1-MxA by using the eukaryotic vector PcDNA3.1 on the basis of obtaining the recombinant adenovirus shuttle plasmid (Adtrack-MxA) containing the target gene MxA. Methods: PAdtrack-MxA and PcDNA3.1 containing the target gene MxA were amplified from Escherichia coli JM109. The recombinant eukaryotic vector PcDNA3.1-MxAwas ligated by using the restriction endonuclease Xba Ignit I for double digestion, and the recombinant eukaryotic vector PcDNA3.1-MxAwas then screened by ampicillin resistance. Double enzyme digestion and PCR identification were used to select the correct clone and sequencing, and DNAssist 2.0 software was used to analyze the homology of the sequence with the previously obtained gene sequence and the known MxA gene sequence in GenBank. Results: the recombinant eukaryotic vector PcDNA3.1-MxA was successfully constructed by restriction endonuclease polymerase chain reaction. The sequencing results showed that the cloned fragment was 2012bpand contained the human MxA gene sequence. The nucleotide sequence analysis showed that the sequence was identical to the target gene sequence obtained in the previous period, but the homology with the human MxA gene sequence in GenBank was 99.75, with only 5 bases different. Only one amino acid encoded changed, which was located in the non-functional region of MxA protein. Conclusion: the recombinant eukaryotic vector PcDNA3.1-MxA was successfully constructed, which laid a foundation for the further study of the anti-hepatitis B virus effect of MxA. The second part of the human MxA gene recombinant eukaryotic vector anti-hepatitis B virus in laboratory study Aim: to study the anti-HBV effect of PcDNA3.1-MxA in vitro and to provide theoretical and experimental basis for further study on anti-virus and clinical application in vivo. Methods: the recombinant eukaryotic vector PcDNA3.1-MxA was transfected into HepG 2.2.15. According to the PcDNA3.1-MxA neomycin resistance, the stable cell clones were screened by G418 resistance. HepG 2.2.15 was divided into experimental group (HepG 2.2.15 with stable expression of PcDNA3.1-MxA) and control group (untransfected group). The RT-PCR method was used to detect the expression of MxA mRNA in HepG2.2.15 and ELISA method was used to detect the level of HBAg-HBeAg in HepG2.2.15. Results: the optimal concentration of G418 was 600 渭 g / ml, and the HepG 2.2.15.RT-PCR amplification of stable expression of PcDNA3.1-MxA at the third week of culture showed that the MxA mRNA level of the experimental group was significantly higher than that of the control group. The results of ELISA test showed that the level of HBsAg HBeAg in the experimental group was significantly lower than that in the control group, and the result was significantly higher than that in the control group (P < 0.01). Conclusion: transfection and stable expression of recombinant eukaryotic vector PcDNA3.1-MxA1MxA in HepG 2.2.15 can inhibit the replication and expression of HBV in HepG 2.2.15.Conclusion: PcDNA3.1-MxA can inhibit the replication and expression of PcDNA3.1-MxA in HepG 2.2.15.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

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